Non-cytotoxic pap mutants

ABSTRACT

Disclosed are PAP mutants that are less toxic than wild type PAP and that exhibit depurination activity. Also disclosed are transgenic plants that procedure the PAP mutants, and methods for preparing the plants. Further disclosed are bioconjugates containing the PAP mutants, pharmaceutical compositions containing the bioconjugates, and methods of administering the compositions to treat disease.

PRIORITY

[0001] This application claims priority on the basis of U.S. provisionalapplication No. 60/266,396, filed Feb. 2, 2001.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] The invention was supported by NSF grant MCB99-82498. Thus, theGovernment may have rights in the invention.

BACKGROUND OF THE INVENTION

[0003] The invention relates to pokeweed antiviral protein andexpression of nucleic acids encoding various PAP mutants in transgenicplants. Many commercially valuable agricultural crops are prone toinfection by plant viruses. These viruses are capable of inflictingsignificant damage to a crop in a given season, and thus can drasticallyreduce its economic value. The reduction in economic value to the farmerin turn results in a higher cost of goods to ultimate purchasers.Several published studies have been directed to the expression of plantvirus capsid proteins in a plant in an effort to confer resistance toviruses. See, e.g., Abel et al., Science 232:738-743 (1986); Cuozzo etal., Bio/Technology 6:549-557 (1988); Hemenway et al., EMBO J.7:1273-1280 (1988); Stark et al., Bio/Technology 7:1257-1262 (1989); andLawson et al., Bio/Technology 8:127-134 (1990). The transgenic plantsexhibited resistance only to the homologous virus and related viruses,however, and not to unrelated viruses. Kawchuk et al., Mol.Plant-Microbe Interactions 3(5):301-307 (1990), disclose the expressionof wild-type potato leafroll virus (PLRV) coat protein gene in potatoplants. Although the infected plants exhibited resistance to PLRV, allof the transgenic plants that were inoculated with PLRV became infectedwith the virus and thus allowed for the continued transmission of thevirus such that high levels of resistance could not be expected. SeeU.S. Pat. No. 5,304,730.

[0004] Pokeweed antiviral protein (PAP) is a 29-kDa Type Iribosome-inhibiting protein (RIP) found in the cell walls of Phytolaccaamericana (pokeweed). See, Wang et al., Adv. Virus Res. 55:325-356(2000). It is a single polypeptide chain that catalytically removes aspecific adenine residue from a highly conserved stem-loop structure(i.e., the α-sarcin loop) in the 28S rRNA of eukaryotic ribosomes, thusinterfering with Elongation Factor-2 binding and blocking cellularprotein synthesis. More specifically, PAP removes an adenine base bycleavage of the N-glycosidic bond at A⁴³²⁴ in rat 28 S rRNA and athomologous sites on ribosomes from other organisms. See, e.g. Irvin etal., Pharmac. Ther. 55:279-302 (1992); Endo et al., Biophys. Res. Comm.150:1032-1036 (1988); and Hartley et al., FEBS Lett. 290:65-68 (1991).PAP recognizes and binds to the ribosomal protein L3 that is essentialfor subsequent depurination of the α-sarcin loop. See, Hudak et al., J.Biol. Chem. 274:3859-3864 (1999).

[0005] PAP protein confers resistance to a broad spectrum of viruseswhen expressed in crop plants, yeast and cultured human cells. Lodge etal., Proc. Natl. Acad. Sci. USA 90:7089-7093 (1993), report theAgrobacterium tumefaciens-mediated transformation of tobacco with a cDNAencoding wild-type pokeweed antiviral protein (PAP) and the resistanceof the transgenic tobacco plants to unrelated viruses. Lodge alsoreports, however, that the PAP-expressing tobacco plants (i.e., above 10ng/mg protein) tended to have a stunted, mottled phenotype, and thatother transgenic tobacco plants that accumulated the highest levels ofPAP were sterile. Since that time, Applicant has found that various PAPmutants provide comparable resistance to plant pests such as viruses andfungi but are less toxic than wild-type PAP. See, U.S. Pat. Nos.5,756,322; 5,880,329 and 6,137,030. The PAP mutants disclosed in theprior art that exhibited less cytotoxicity (e.g., phytotoxicity) thanwild-type PAP also exhibited the capability of depurinating the cellribosomes. The belief was that cytotoxic effect was a result oftranslation inhibition due to depurinated rRNA.

SUMMARY OF THE INVENTION

[0006] Applicants have discovered that PAP depurination can occur in theabsence of cytotoxicity and that both events are independent. That is,just because a PAP protein depurinates a ribosome and thus caneffectively interfere with the ability of a cell to manufacture proteinsdoes not mean the PAP protein will also be cytotoxic.

[0007] One aspect of the present invention is directed to PAP mutantsthat depurinate the ribosomes of the cell, but are less toxic to cellsthan wild type PAP. The Pokeweed Antiviral Protein (PAP) mutant is saidto be substantially non-toxic and exhibits ribosome depurinationactivity. One preferred PAP mutant differs from wild-type PAP in thatthe native tyrosine residue at position 123 is replaced by alanine(hereinafter PAP (1-262, Y123A)). Other preferred PAP mutants containPAP (1-262, S14M, Y16A), PAP (1-262, L71R), PAP (1-262, V73E), PAP(1-262, M74R), PAP (1-262, Y76A) and PAP (1-262, Y1231). Yet otherpreferred PAP mutants differ from wild-type PAP substantially in thatthey are truncated at their C-termini from 10 to 20 mature PAP aminoacids. These PAP mutants are designated PAP (1-242), PAP (1-243), PAP(1-244), PAP (1-245), PAP (1-246), PAP (1-247), PAP (1-248), PAP(1-249), PAP (1-250) and PAP (1-251). DNAs encoding the PAP mutants,chimeric constructs thereof, including vectors and non-human hoststransformed with the constructs, are also provided.

[0008] Another aspect of the present invention is directed to transgenicplants that express nucleic acids encoding the PAP mutants. The plantsexhibit resistance to a broad spectrum of plant pests such as virusesand fungi. The invention also provides plant parts e.g., leaves, stemsand shoots, as well as plant cells and protoplasts, containing a DNAmolecule encoding a PAP mutant, from which whole plants expressing theDNA are generated. The invention applies to flowering plants in general,including both monocots and dicots. In preferred embodiments, the plantsare corn, rice, wheat, turfgrass, soybean, cotton canola, potato, tomatoand cucurbits. Seed derived from the transgenic plants is also provided.Methods of making the transgenic plants are further provided.

[0009] The PAP mutants of the present invention also have utility asbiotherapeutic agents. Thus, a further aspect of the present inventionis directed to a fusion protein or an immunoconjugate containing the PAPmutant and a targeting moiety that binds a receptor on or in a cell. Thetargeting moiety is a ligand that specifically targets to infected,diseased or otherwise unwanted cells. Thus, methods of treating mammalse.g., humans, suffering from diseases characterized by the presenceand/or abnormal growth of such cells are also provided. In preferredembodiments, the agent is designed to target cells infected with avirus, or a cancer cell. DNAs encoding the fusion proteins, andconstructs containing the DNAs, therapeutic compositions containing thefusion proteins or immunoconjugates, and methods of using same e.g., totreat cancer or AIDS patients, are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 is a graph showing growth of yeast cells that expresswild-type PAP (PAPwt) or PAP mutants of the present invention.

[0011]FIGS. 2A, 2B and 2C are bar graphs showing depurination activityof wild-type PAP (PAPwt) and two PAP mutants of the present inventionrespectively, that are expressed in yeast cells.

[0012]FIGS. 3A and 3B are bar graphs showing effect of expression of aPAP mutant of the present invention and PAPwt respectively, onproduction of their mRNA, as a function of time.

[0013]FIG. 4 is a graph showing depurination activity of a PAP mutant ofthe present invention PAPL71R (NT538) and PAPwt (NT188).

[0014]FIGS. 5A and B are bar graphs showing the effect of expression ofa PAP mutant of the present invention, PAP L71R (NT538) and PAPwt(NT188) respectively, on the stability of their mRNA

[0015]FIG. 6A (1 and 2) are photographs of plates showing serialdilution of yeast cells growing on media containing glucose, wherein theyeast express PAPwt (1) and a PAP mutant of the present invention PAPL71R (NT538) (2).

[0016]FIG. 6B is a bar graph showing the quantification of the viabilityassay and a comparison of yeast colony forming units at indicated timesafter induction of expression of a PAP mutant of the present inventionPAPL71R (NT538) and PAP wt.

[0017]FIG. 6C is a graph showing growth of yeast that produce a PAPmutant of the present invention, yeast that produce the nontoxic PAPmutant (E176V) and yeast that produce wild type PAP.

BEST MODE OF CARRYING OUT THE INVENTION

[0018] By “wild-type PAP,” it is meant the PAP amino acid sequence1-262, the 22-amino acid N-terminal signal peptide (“the N-terminalsignal sequence of wild-type PAP”), and the 29 amino acid C-terminalextension (amino acids enumerated 263-291), set forth below as SEQ IDNO:2. The corresponding nucleotide sequence is set forth as SEQ ID NO:1.Thus, by the terms “wild-type, mature PAP,” or “mature PAP”, it is meantthe PAP amino acid sequence 1-262.5′CTATGAAGTCGGGTCAAAGCATATACAGGCTATGCATTGTTAGAAACATTGATGCCTCTGATCCCGATAAACAATACAAATTAGACAATAAGATGACATACAAGTACCTAAACTGTGTATGGGGGAGTGAAACCTCAGCTGCTAAAAAAACGTTGTAAGAAAAAAAGAAAGTTGTGAGTTAACTACAGGGCGAAAGTATTGGAACT AGCTAGTAGGAAGGGAAG ATG AAGTCG ATG CTT GTG GTG ACA ATA TCA ATA                     Met Lys Ser MetLeu Val Val Thr Ile Ser Ile                                         (67)TGG CTC ATT CTT GCA CCA ACT TCA ACT TGG GCT GTG AAT ACA ATC ATC TAC TrpLeu Ile Leu Ala Pro Thr Ser Thr Trp Ala Val Asn Thr Ile Ile Tyr                                            (1)                 (100)AAT GTT GGA AGT ACC ACC ATT AGC AAA TAC GCC ACT TTT CTG AAT GAT CTT AsnVal Gly Ser Thr Thr Ile Ser Lys Tyr Ala Thr Phe Leu Asn Asp Leu            (10)                                (20) CGT AAT GAA GCG AAAGAT CCA AGT TTA AAA TGC TAT GGA ATA CCA ATG CTG Arg Asn Glu Ala Lys AspPro Ser Leu Lys Cys Tyr Gly Ile Pro Met Leu                    (30)                                    (40) CCC AATACA AAT ACA AAT CCA AAG TAC GTG TTG GTT GAG CTC CAA GGT TCA Pro Asn ThrAsn Thr Asn Pro Lys Tyr Val Leu Val Glu Leu Gln Gly Ser                                (50) AAT AAA AAA ACC ATC ACA CTA ATG CTGAGA CGA AAC AAT TTG TAT GTG ATG Asn Lys Lys Thr Ile Thr Leu Met Leu ArgArg Asn Asn Leu Tyr Val Met        (60)                                (70) GGT TAT TCT GAT CCC TTTGAA ACC AAT AAA TGT CGT TAC CAT ATC TTT AAT Gly Tyr Ser Asp Pro Phe GluThr Asn Lys Cys Arg Tyr His Ile Phe Asn                    (80)                                (90) GAT ATC TCAGGT ACT GAA CGC CAA GAT GTA GAG ACT ACT CTT TGC CCA AAT Asp Ile Ser GlyThr Glu Arg Gln Asp Val Glu Thr Thr Leu Cys Pro Asn                            (100) GCC AAT TCT CGT GTT ACT AAA AAC ATAAAC TTT GAT AGT CGA TAT CCA ACA Ala Asn Ser Arg Val Ser Lys Asn Ile AsnPhe Asp Ser Arg Tyr Pro Thr    (110)                               (120) TTG GAA TCA AAA GCG GGAGTA AAA TCA AGA AGT CAG GTC CAA CTG GGA ATT Leu Glu Ser Lys Ala Gly ValLys Ser Arg Ser Gln Val Gln Leu Gly Ile                (130)                               (140) CAA ATA CTCGAC AGT AAT ATT GGA AAG ATT TCT GGA GTG ATG TCA TTC ACT Gln Ile Leu AspSer Asn Ile Gly Lys Ile Ser Gly Val Met Ser Phe Thr                        (150) GAG AAA ACC GAA GCC GAA TTC CTA TTG GTAGCC ATA CAA ATG GTA TCA GAG Glu Lys Thr Glu Ala Glu Phe Leu Leu Val AlaIle Gln Met Val Ser Glu (160)                               (170) GCAGCA AGA TTC AAG TAC ATA GAG AAT CAG GTG AAA ACT AAT TTT AAC AGA Ala AlaArg Phe Lys Tyr Ile Glu Asn Gln Val Lys Thr Asn Phe Asn Arg            (180)                               (190) GCA TTC AAC CCTAAT CCC AAA GTA CTT AAT TTG CAA GAG AGA TGG GGT AAG Ala Phe Asn Pro AsnPro Lys Val Leu Asn Leu Gln Glu Thr Trp Gly Lys                        (200)                           (210) ATT TCAACA GCA ATT CAT GAT GCC AAG AAT GGA GTT TTA CCC AAA CCT CTC Ile Ser ThrAla Ile His Asp Ala Lys Asn Gly Val Leu Pro Lys Pro Leu                                (220) GAG CTA GTG GAT GCC AGT GGT GCCAAG TGG ATA GTG TTG AGA GTG GAT GAA Glu Leu Val Asp Ala Ser Gly Ala LysTrp Ile Val Leu Arg Val Asp Glu        (230)                               (240) ATC AAG CCT GAT GTAGCA CTC TTA AAC TAC GTT GGT GGG AGC TGT CAG ACA Ile Lys Pro Asp Val AlaLeu Leu Asn Tyr Val Gly Gly Ser Cys Gln Thr                    (250)                               (260) ACT TATAAC CAA AAT GCC ATG TTT CCT CAA CTT ATA ATG TCT ACT TAT TAT Thr Tyr AsnGln Asn Ala Met Phe Pro Gln Leu Ile Met Ser Thr Tyr Tyr(262)                       (270) AAT TAC ATG GTT AAT CTT GGT GAT CTATTT GAA GGA TTC TGATCATAAACA Asn Tyr Met Val Asn Leu Gly Asp Leu Phe GluGly Phe     (280)                               (290)TAATAAGGAGTATATATATATTACTCCAACTATATTATAAAGCTTAAATAAGAGGCCGTGTTAATTAGTACTTGTTGCCTTTTGCTTTATGGTGTTGTTTATTATGCCTTGTATGCTTGTAATATTATCTAGAGAACAAGATGTACTGTGTAATAGTCTTGTTTGAAATAAAACTTCCAATTATGATGCAAAAAAAAAAAAAAA3′

[0019] The sequences contain 5′ and 3′ non-coding, flanking sequences.Upon expression in eukaryotic cells, the N-terminal 22-amino acidsequence of wild-type PAP is co-translationally cleaved, yielding apolypeptide having a molecular weight of about 32 D, which is thenfurther processed by the cleavage of the C-terminal 29-amino acids (“theC-terminal extension of wild-type PAP” or “PAP (263-292)”), yieldingmature, wild-type PAP (hereinafter “PAP (1-262)”) (i.e., that which isisolated from Phytolacca americana leaves), having a molecular weight ofabout 29 kD. See Irvin et al., Pharmac. Ther. 55:279-302 (1992); Dore etal., Nuc. Acids Res. 21(18):4200-05 (1993); Monzingo et al., J. Mol.Biol. 233:705-15 (1993); and Turner et al., Proc. Natl. Acad. Sci. USA92:8448-52 (1995). PAP has been further characterized in terms of threedistinct domains, namely the N-terminal domain which includes amino acidresidues 1-69, a central domain which includes amino acid residues70-179 and a C-terminal domain which includes amino acid residues180-262. The PAP mutants embraced by the present invention includeN-terminal domain mutants, central domain mutants and C-terminal domainmutants.

[0020] The terms “depurination”, “depurination activity” or“depurination catalytic activity” are used interchangeably herein. Theseterms are not interpreted so as to require depurination of adeninylresidues from ribosomal RNA at the same rate and/or to the same extentas achieved by wild-type PAP. For example, the extent of depurinationmay be comparable to or even exceed that of wild-type PAP over a giventime period. Differences in the rate and/or extent of depurinationactivity of preferred PAP mutants of the present invention compared towild-type PAP are graphically illustrated in FIGS. 2B, 2C and 4. On theother hand, the depurination activity is non-negligible. An example of aPAP mutant that exhibits negligible depurination activity is the activesite PAP mutant E176V. Methods for measuring depurination activityinvolving primer extension are described in the examples. Another methodsuitable for this purpose involves an RNase protection assay. RNA isisolated from the cell that expresses the given PAP mutant in accordancewith standard techniques. It is cleaved at the site of depurination(e.g., such as by treatment with aniline). An RNase protection probe isdesigned, based on nucleotides that flank the depurination site. Tosynthesize the probe, forward and reverse primers complementary to rRNAsequences that flank the depurination site may be prepared, which inturn will generate a PCR product using cellular (e.g., yeast) RNA as atemplate. One of the primers contains a promoter (e.g., phage T7polymerase promoter) such that it is incorporated into the PCR productThe product is then used in an RNase protection assay (preferably asdescribed in Tumer et al., J. Virol. 72:1036-1042 (1998)). Once thedepurinated rRNA is cleaved, the probe will yield protected fragments ofdifferent sizes, which can be visualized on a sequencing gel.

[0021] The PAP mutants of the present invention are less cytotoxic thanwild-type PAP. By less cytotoxic or toxic, it is meant that the PAPmutants do not significantly inhibit cell growth like wild type PAP butin any event do not significantly affect cell viability. Thisdetermination can be made in accordance with a combination of standardtechniques, illustrations of which are set forth in the examples. First,a determination is made as to whether the PAP mutant inhibits cellgrowth as compared to wild type PAP. One method involves growing cellssuch as yeast that produce the PAP mutant in question and plating andre-plating the cells on selective media. Another method involvesmeasuring doubling time of growth of the cells in selective media afterthe induction of PAP production. As can be seen from example 4 (and theresults shown in table 3), PAP mutants exhibiting doubling timesapproximating the doubling time for wild type PAP tended to be toxic andthus outside the scope of the present invention. PAP mutants that causeddoubling times of cells approximately PAPx (the active site mutant,E176V), tended to be nontoxic. The second step involves a determinationof whether the PAP mutant causes cell death. In this case, a viabilityassay will distinguish between PAP mutants that cause cell death andthat are toxic versus PAP mutants that might appear to be toxic (onaccount of having a doubling time approximately that for wild type PAP)but actually do not cause cell death, and thus are considered to benontoxic. There are also unusual situations in which a PAP mutantappears to be nontoxic based on its effect on cell growth but is stilltoxic. (See table 3 and comparative data for S14M versus S14M, Y16A.)These phenomena may be explained by the fact that inhibition of cellgrowth does not always correlate with cell viability. Thus, regardlessof whether the PAP mutants of the present invention have a significanteffect on doubling time of cells, their effect on cell viability issignificantly less than that of wild-type PAP.

[0022] Rajamohan et al., J. Biol. Chem. 275(5):3382-90 (2000), report aPAP mutant having a double alanine substitution at residues 122 and 123(i.e., R122A, Y123A, in Applicants' nomenclature) resulted in nearlycomplete loss (i.e., greater than 1700-fold less active) of ribosomaldepurination activity. The working hypothesis is that Y123 is an activesite residue and thus plays an important role in catalyticdeadenylation. In direct contrast to these results, one of Applicants'preferred embodiments, a PAP mutant containing an active sitesubstitution at position Y123, i.e., PAP (1-262, Y123A), exhibitsdepurination activity similar to that of wild-type PAP. As shown inexample 1, by 8 hours post induction, similar levels of depurinationwere observed in the wild-type PAP and in the Y123A mutant PAP (NT242).The alanine residue at position 123 of a preferred PAP mutant is a morepreferred substitution; it is not critical. That is, other substitutionse.g., conservative substitutions with other neutral amino acids, i.e.,glycine, valine, isoleucine, leucine, proline and methionine can bemade. Other preferred PAP mutants contain PAP (1-262, S14M, Y16A), PAP(1-262, L71R), PAP (1-262, V73E), PAP (1-262, M74R), PAP (1-262, Y76A)and PAP (1-262, Y123I). In the case of the single substitution mutants,other amino acid residues can be substituted, preferably in aconservative manner, provided that the requisite properties aremaintained. In the case of the double substitution mutant, the S14Mmutation is important but amino acid residues other than alanine may besubstituted for Y at position 16 provided once again, that the requisiteproperties are maintained.

[0023] Yet other preferred PAP mutants differ from wild-type PAPsubstantially in that they are truncated at their C-termini from 10 to20 mature PAP amino acids. These PAP mutants are designated PAP (1-242),PAP (1-243), PAP (1-244), PAP (1-245), PAP (1-246), PAP (1-247), PAP(1-248), PAP (1-249), PAP (1-250), PAP (1-251) and PAP (1-252).

[0024] Yet other PAP mutants of the present invention may be identifiedby random mutagenesis of PAP-encoding nucleic acids, expression of themutagenized nucleic acids in cells, and testing the mutants fordepurination and toxicity. Preferred cells in which to identifynon-toxic PAP mutants are yeast cells. Once non-toxic, depurinating PAPmutants are identified, the nucleic acids may be sequenced.

[0025] Expression of the PAP mutants of the present invention in atransgenic plant confers broad spectrum virus resistance, i.e.,resistance to or the capability of suppressing infection by a number ofunrelated viruses, including but not limited to RNA viruses e.g.potexviruses such as (PVX, potato virus X), potyvirus (PVY), cucumbermosaic virus (CMV), tobacco mosaic viruses (TMV), barley yellow dwarfvirus (BYDV), wheat streak mosaic virus, potato leaf roll virus (PLRV),plumpox virus, watermelon mosaic virus, zucchini yellow mosaic virus,papaya ringspot virus, beet western yellow virus, soybean dwarf virus,carrot read leaf virus and DNA plant viruses such as tomato yellow leafcurl virus. See also Lodge et al., supra., Tomlinson et al., J. Gen.Virol. 22:225-32 (1974); and Chen et al., Plant Pathol. 40:612-20(1991).

[0026] Expression of the PAP mutants in plants also confers resistanceto a broad spectrum of fungi that infect plants. There is increasedresistance to diseases caused by plant fungi, including those caused byPythium (one of the causes of seed rot, seedling damping off and rootrot), Phytophthora (the cause of late blight of potato and of root rots,and blights of many other plants), Bremia, Peronospora, Plasmopara,Pseudoperonospora and Sclerospora (causing downy mildews), Erysiphegraminis (causing powdery mildew of cereals and grasses), Verticillium(causing vascular wilts of vegetables, flowers, crop plants and trees),Rhizoctonia (causing damping off disease of many plants and brown patchdisease of turfgrasses), Fusarium (causing root rot of bean, dry rot ofpotatoes), Cochliobolus (causing root and foot rot, and also blight ofcereals and grasses), Giberella (causing seedling blight and foot orstalk rot of corn and small grains), Gaeumannomyces (causing thetake-all and whiteheads disease of cereals), Schlerotinia (causing crownrots and blights of flowers and vegetables and dollar spot disease ofturfgrasses), Puccinia (causing the stem rust of wheat and other smallgrains), Ustilago (causing corn smut), Magnaporthae (causing summerpatch of turfgrasses), and Schlerotium (causing southern blight ofturfgrasses). Other important fungal diseases include those caused byCercospora, Septoria, Mycosphoerella, Gloinerella, Colletotrichum,Helminthosporium, Alterneria, Botrytis, Cladosporium and Aspergillus.Applicant also believes that the PAP mutants confer increased resistanceto insects, bacteria and nematodes in plants. Important bacterialdiseases to which the PAP mutants impart increased resistance includethose caused by Pseudomonas, Xanthomonas, Erwinia, Clavibacter andStreptomyces.

[0027] DNAs encoding the PAP mutants can be made in accordance withstandard techniques. See Ausubel et al. (eds.), Vol. 1, Chap. 8 inCurrent Protocols in Molecular Biology, Wiley, N.Y. (1990). The DNAs mayalso be prepared via PCR techniques. See PCR Protocols, Innis et al.(eds.), Academic Press, San Diego, Calif. (1990). Referring back to theamino acid and corresponding nucleotide sequences of wild type PAP, forexample, the codon changes at the nucleotide level for theaforementioned N-terminal domain and central domain mutants are asfollows: PAP (1-262, S14M, Y16A) (codon for S, AGC, changed to ATG, andthe codon for Y, TAC, changed to GCC); PAP (1-262, L71R) (codon for L,TTG, changed to CGG); PAP (1-262, V73#) (codon for V, GTG, changed toGAG); PAP (1-262, M74R) (codon for M, ATG, changed to CGG); PAP (1-262,Y76A) (codon for Y, TAT, changed to GCT); PAP (1-262, Y123A) (codon forY, TAT, changed to GCT); and PAP (1-22, Y123I) (codon for Y, TAT,changed to ATT). The PAP DNA (e.g., a cDNA) is preferably inserted intoa plant transformation vector in the form of an expression cassettecontaining all of the necessary elements for transformation of plantcells. The expression cassette typically contains, in proper readingframe, a promoter functional in plant cells, a 5′ non-translated leadersequence, the mutant PAP DNA, and a 3′ non-translated region functionalin plants to cause the addition of polyadenylated nucleotides to the 3′end of the RNA sequence. Promoters functional in plant cells may beobtained from a variety of sources such as plants or plant DNA viruses.The selection of a promoter used in expression cassettes will determinethe spatial and temporal expression pattern of the construction in thetransgenic plant. Selected promoters may have constitutive activity andthese include the CaMV 35S promoter, the actin promoter (McElroy et al.Plant Cell 2:163-71 (1990); McElroy et al. Mol. Gen. Genet. 231:150-160(1991); Chibbar et al. Plant Cell Rep. 12:506-509 (1993), and theubiquitin promoter (Binet et al. Plant Science 79:87-94 (1991),Christensen et al. Plant Mol. Biol. 12:619-632 (1989); Taylor et al.Plant Cell Rep. 12:491-495 (1993)). Alternatively, they may be induciblee.g., wound-induced (Xu et al., Plant Mol. Biol 22:573-588 (1993),Logemann et al., Plant Cell 1:151-158 (1989), Rohrmeier & Lehle, PlantMol. Bio. 22:783-792 (1993), Firek et al. Plant Mol. Biol. 22:129-142(1993), Warener et al. Plant J. 3:191-201 (1993)) and thus drive theexpression of the mutant PAP gene at the sites of wounding or pathogeninfection. Other useful promoters are expressed in specific cell types(such as leaf epidermal cells, meosphyll cells, root cortex cells) or inspecific tissues or organs (roots, leaves or flowers, for example).Patent Application WO 93/07278, for example, describes the isolation ofthe maize trpA gene that is preferentially expressed in pith cells.Hudspeth et al., Plant Mol. Biol. 12:579-589 (1989), have described apromoter derived from the maize gene encoding phosphoenolpyruvatecarboxylase (PEPC) which directs expression in a leaf-specific manner.Alternatively, the selected promoter may drive expression of the geneunder a light induced or other temporally regulated promoter. A furtheralternative is that the selected promoter be chemically regulated.

[0028] A variety of transcriptional cleavage and polyadenylation sitesare available for use in expression cassettes. These are responsible forcorrect processing (formation) of the 3′ end of mRNAs. Appropriatetranscriptional cleavage and polyadenylation sites functional in plantsinclude the CaMV 35S cleavage and polyadenylation sites, the tmlcleavage and polyadenylations sites, the nopaline synthase cleavage andpolyadenylation sites, the pea rbcS E9 cleavage and polyadenylationsites. These can be used in both monocotyledons and dicotyledons.

[0029] Numerous sequences have been found to enhance gene expressionfrom within the transcriptional unit and these sequences can be used inconjunction with the genes of this invention to increase theirexpression in transgenic plants. Various intron sequences have beenshown to enhance expression, particularly in monocotyledonous cells. Forexample, the introns of the maize Adhl gene have been found tosignificantly enhance the expression of the wild-type gene under itscognate promoter when introduced into maize cells. Intron 1 was found tobe particularly effective and enhanced expression in fusion constructswith the chloramphenicol acetyltransferase gene (Callis et al., GenesDevelop 1:1183-1200 (1987)). In the same experimental system, the intronfrom the maize bronze-l gene had a similar effect in enhancingexpression (Callis et al., supra.). Intron sequences have been routinelyincorporated into plant transformation vectors, typically within thenon-translated leader. A number of non-translated leader sequencesderived from viruses are also known to enhance expression, and these areparticularly effective in dicotyledonous cells. Specifically, leadersequences from Tobacco Mosaic Virus (TMV, the “Ω-sequence”), MaizeChlorotic mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have beenshown to be effective in enhancing expression (e.g. Gallie et al. Nucl.Acids Res. 15:8693-8711 (1987); Skuzeski et al. Plant Mol. Biol.15:65-79 (1990)).

[0030] Numerous transformation vectors are available for planttransformation, and the genes of this invention can be used inconjunction with any such vectors. The selection of vector for use willdepend upon the preferred transformation technique and the targetspecies for transformation. For certain target species, differentantibiotic or herbicide selection markers may be preferred. Selectionmarkers used routinely in transformations include the nptII gene whichconfers resistance to kanamycin (Messing et al., Gene 19:259-268 (1982);Bevan et al., Nature 304:184-187 (1983)), the bar gene which confersresistance to the herbicide phosphinothricin (White et al., Nucl. AcidsRes. 18:1062 (1990); Spencer et al., Theor. Appl. Genet. 79:625-631(1990)), the hph gene which confers resistance to the antibiotichygromycin, and the dhfr gene, which confers resistance to methotrexate.Vectors suitable for Agrobacterium transformation typically carry atleast one T-DNA border sequence. These include vectors such as pBIN19and pCIB200 (EP 0 332 104).

[0031] Transformation without the use of Agrobacterium tumefacienscircumvents the requirement for T-DNA sequences in the chosentransformation vector and consequently vectors lacking these sequencescan be utilized in addition to vectors such as the ones described abovewhich contain T-DNA sequences. Transformation techniques which do notrely on Agrobacterium include transformation via particle bombardment,protoplast uptake (e.g., PEG and electroporation) and microinjection.The choice of vector depends largely on the preferred selection for thespecies being transformed. For example, pCIB3064 is a pUC-derived vectorsuitable for the direct gene transfer technique in combination withselection by the herbicide basta (or phosphinothricin). It is describedin WO 93/07278 and Koziel et al. (Biotechnology 11:194-200 (1993)).

[0032] An expression cassette containing the mutant PAP gene DNAcontaining the various elements described above may be inserted into aplant transformation vector by standard recombinant DNA methods.Alternatively, some or all of the elements of the expression cassettemay be present in the vector, and any remaining elements may be added tothe vector as necessary.

[0033] Transformation techniques for dicotyledons are well known in theart and include Agrobacterium-based techniques and techniques which donot require Agrobacterium. Non-Agrobacterium techniques involve theuptake of exogenous genetic material directly by protoplasts or cells.This can be accomplished by PEG or electroporation mediated uptake,particle bombardment-mediated delivery, or microinjection. Examples ofthese techniques are described by Paszkowski et al., EMBO J. 3:2717-2722(1984), Potrykis et al., Mol. Gen. Genet. 199:169-177 (1985), Reich etal., Biotechnology 4:1001-1004 (1986), and Klein et al., Nature327:70-73 (1987). In each case the transformed cells are regenerated towhole plants using standard techniques.

[0034]Agrobacterium-mediated transformation is a preferred technique fortransformation of dicotyledons because of its high efficiency oftransformation and its broad utility with many different species. Themany crop species which are routinely transformable by Agrobacteriuminclude tobacco, tomato, sunflower, cotton, oilseed rape, potato,soybean, alfalfa and poplar (EP 0 317 511 (cotton), EP 0 249 432(tomato), WO 87/07299 (Brassica), U.S. Pat. No. 4,795,855 (poplar)).Agrobacterium transformation typically involves the transfer of thebinary vector carrying the foreign DNA of interest (e.g. pCIB200 orpCIB2001) to an appropriate Agrobacterium strain which may depend on thecomplement of vir genes carried by the host Agrobacterium strain eitheron a co-resident plasmid or chromosomally (e.g. strain CIB542 forpCIB200 (Uknes et al. Plant Cell 5:159-169 (1993)). The transfer of therecombinant binary vector, to Agrobacterium is accomplished by atriparental mating procedure using E. coli carrying the recombinantbinary vector, a helper E. coli strain which carries a plasmid such aspRK2013 which is able to mobilize the recombinant binary vector to thetarget Agrobacterium strain. Alternatively, the recombinant binaryvector can be transferred to Agrobacterium by DNA transformation (Höfgen& Willmitzer, Nucl. Acids Res. 16:9877 (1988)).

[0035] Transformation of the target plant species by recombinantAgrobacterium usually involves co-cultivation of the Agrobacterium withexplants from the plant and follows protocols known in the art.Transformed tissue is regenerated on selectable medium carrying anantibiotic or herbicide resistance marker present between the binaryplasmid T-DNA borders. Preferred transformation techniques for monocotsinclude direct gene transfer into protoplasts using PEG orelectroporation techniques and particle bombardment into callus tissue.Transformation can be undertaken with a single DNA species or multipleDNA species (i.e. co-transformation) and both these techniques aresuitable for use with this invention. Co-transformation may have theadvantage of avoiding complex vector construction and of generatingtransgenic plants with unlinked loci for the gene of interest and theselectable marker, enabling the removal of the selectable marker insubsequent generations, should this be regarded desirable. However, adisadvantage of the use of co-transformation is the less than 100%frequency with which separate DNA species are integrated into the genome(Schocher et al., Biotechnology 4:1093-1096 (1986)). Published PatentApplications EP 0 292 435, EP 0 392 225 and WO 93/07278 describetechniques for the preparation of callus and protoplasts of maize,transformation of protoplasts using PEG or electroporation, and theregeneration of maize plants from transformed protoplasts. Gordeon-Kammet al., Plant Cell 2:603-618 (1990), and Fromm et al., Biotechnology11:194-200 (1993), describe techniques for the transformation of eliteinbred lines of maize by particle bombardment.

[0036] Transformation of rice can also be undertaken by direct genetransfer techniques utilizing protoplasts or particle bombardmentProtoplast-mediated transformation has been described for Japonica-typesand Indica-types (Zhange et al., Plant Cell Rep. 7:739-384 (1988);Shimamoto et al. Nature 338:274-277 (1989); Datta et al. Biotechnology8:736-740 (1990)). Both types are also routinely transformable usingparticle bombardment (Christou et al. Biotechnology 9:957-962 (1991)).

[0037] Patent Application EP 0 332 581 described techniques for thegeneration, transformation and regeneration of Pooideae protoplasts.Furthermore wheat transformation has been described by Vasil et al.,Biotechnology 10:667-674 (1992), using particle bombardment into cellsof type C long-term regenerable callus, and also by Vasil et al.,Biotechnology 11:1553-1558 (1993), and Weeks et al., Plant Physiol.102:1077-1084 (1993), using particle bombardment of immature embryos andimmature embryo-derived callus.

[0038] Transformation of monocot cells such as Zea mays can be achievedby bringing the monocot cells into contact with a multiplicity ofneedle-like bodies on which these cells may be impaled, causing arupture in the cell wall thereby allowing entry of transforming DNA intothe cells. See U.S. Pat. No. 5,302,523. Transformation techniquesapplicable to both monocots and dicots are also disclosed in thefollowing U.S. Pat. No. 5,240,855 (particle gun); U.S. Pat. No.5,204,253 (cold gas shock accelerated microprojectiles); U.S. Pat. No.5,179,022 (biolistic apparatus); U.S. Pat. Nos. 4,743,548 and 5,114,854(microinjection); and U.S. Pat. Nos. 5,149,655 5,120,657 (acceleratedparticle mediated transformation); U.S. Pat. No. 5,066,587 (gas drivenmicroprojectile accelerator); U.S. Pat. No. 5,015,580 (particle-mediatedtransformation of soy bean plants); U.S. Pat. No. 5,013,660 (laserbeam-mediated transformation); and U.S. Pat. Nos. 4,849,355 and4,663,292.

[0039] The transformed plant cells or plant tissue are then grown intofull plants in accordance with standard techniques. Transgenic seed canbe obtained from transgenic flowering plants in accordance with standardtechniques. Likewise, non-flowering plants such as potato and sugarbeets can be propagated by a variety of known procedures. See e.g.Newell et al., Plant Cell Rep. 10:30-34 (1991) (disclosing potatotransformation by stem culture).

[0040] The PAP mutants confer resistance to a broad spectrum of fungaland/or viral diseases to plants. Examples of such plants are floweringplants including monocots (e.g., cereal crops) and dicots. Specificexamples include maize, tomato, turfgrass, asparagus, papaya, sunflower,rye, beans, ginger, lotus, bamboo, potato, rice, peanut, barley, malt,wheat, alfalfa, soybean, oat, eggplant, squash, onion, broccoli,sugarcane, sugar beet, beets, apples, oranges, grapefruit, pear, plum,peach, pineapple, grape, rose, carnation, daisy, tulip, Douglas fir,cedar, white pine, scotch pine, spruce, peas, cotton, flax and coffee.As an alternative to preparing transgenic plants containing an exogenousmutant PAP gene (or a PAP transgene), the PAP mutant protein may beapplied directly onto the plants.

[0041] Another aspect of the present invention is directed tobioconjugates containing the PAP mutants. Fusion proteins are hybridproteins containing a polypeptide toxin and a targeting moiety made byrecombinant DNA technology. Thus, the toxic and targeting moieties arejoined together via a peptide bond. See, U.S. Pat. Nos. 4,675,382 and5,616,482 (the “'382 and the '482 patents”). Immunotoxins on the otherhand are not fusion proteins; in these cases, the toxic moiety and thetargeting moieties are linked via a non-peptide bond. Thus, in the caseof immunotoxins, the targeting moiety does not have to be peptidic innature. The targeting moiety will cause binding of the immunotoxin orfusion protein (collectively “bioconjugate”) to a target cell such as acancer cell, a T-cell, monocyte or macrophage. Thus, in its broadestcontext, the bioconjugates of the present invention may be used totarget any unwanted cell to which it will bind. The targeting moiety isselected as to preferentially bind to the target cells to thesubstantial exclusion of non-target (e.g., non-diseased) cells becausethe receptor or binding partner that binds the targeting moiety is notpresent on or in the target cell, or is present but in significantlyfewer numbers so as to reduce unwanted side effects.

[0042] The bioconjugates in the form of an immunotoxin may be preparedby a process involving preparing the recombinant PAP by (i) cloning aDNA sequence that encodes the PAP mutant into an expression vector; (ii)transforming E. coli cells or other host cells with the expressionvector; (iii) maintaining the transformed cells under biologicconditions sufficient for expression of the PAP mutant. Once therecombinant PAP mutant has been isolated, a suitable targeting moiety isprovided such as a monoclonal antibody, monoclonal antibody fragment, ora single chain variable region polypeptide in purified form. The PAPmutant is then linked to the targeting moiety by methods of conjugationwell known to those of skill in the art. For example, one such method isthe utilization of a heterobifunctional crosslinker, e.g. N-succinimidyl3-(2-pyridyldithio)propionate (SPDP). Finally, the immunotoxin ispurified by size exclusion and affinity chromatography and any endotoxinis removed. A method of linking B43 to a toxic moiety (e.g., a PAPmutant of the present invention) is taught in U.S. Pat. No. 4,831,117.Yet other methods of linking the targeting moiety to the PAP mutant aretaught in U.S. Pat. Nos. 4,363,758; 5,167,956 and 4,340,535. In additionto SPDP, 4-succinimidyloxycarbonyl-methyl-(2-pyridyldithio)-toluene(SMPT) and N-succimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate(LC-SPDP) are commonly used as linking agents.

[0043] Also provided in the present invention is a process for preparinga fusion toxin. Typically, the process involves constructing anexpression vector comprising DNA encoding a fusion protein comprisingthe PAP mutant and the targeting moiety. The vector is utilized in thetransformation of a non-human host such as E. coli cells. Thetransformed cells are then maintained under biologic conditionssufficient for expression of the fusion toxin. Once the fusion toxin hasbeen expressed, it can be isolated and purified by size exclusion andaffinity chromatography steps, and any endotoxin can be removed.

[0044] The general methods for constructing recombinant DNA which cantransform target cells are well known to those skilled in the art, andthe same compositions and methods of construction may be utilized toproduce the recombinant PAP useful herein. For example, J. Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press (2d ed., 1989), provides suitable methods ofconstruction.

[0045] The recombinant nucleic acid can be readily introduced into thetarget cells by transfection with an expression vector comprising cDNAencoding PAP, for example, by the modified calcium phosphateprecipitation procedure of C. Chen et al., Mol. Cell. Biol., 7:2745(1987). Transfection can also be accomplished by lipofectin, usingcommercially available kits, e.g., provided by BRL. Suitable non-humanhosts include cells for the expression of the recombinant PAP derivedfrom multicellular organisms, such as yeasts, insects and plants. Suchhosts are capable of complex processing and glycosylation activities. Inprinciple, any higher eukaryotic cell culture is functional, whetherfrom vertebrate or invertebrate culture. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyxmori have been identified. See, e.g., Luckow et al., Bio/Technol, 6:47(1988); Miller et al., in Genetic Engineering, J. K. Setlow et al.,eds., Vol. 8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al.,Nature, 315:592 (1985). A is variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may beused, preferably for transfection of Spodoptera frugiperda cells.

[0046] In preferred embodiments, the bioconjugates are used for thetreatment of cancer. The method comprises parenterally administering toa patient who is afflicted with cancer an effective amount of apharmaceutical composition comprising a bioconjugate of the presentinvention. Preferred cancers are diseases associated with theproliferation of mammalian cells expressing antigens recognized by thepreferred targeting moieties of the present invention. Such cancersinclude, but are not limited to, B-lineage acute lymphoblastic leukemia,chronic lymphocytic leukemia, B-lineage lymphoma, blast crisis ofchronic myelocytic leukemia, hairy cell leukemia, AIDS lymphoma,EBV-lymphoma, brain tumors, neuroblastoma, osteosarcoma, soft tissuesarcoma, breast cancer, prostate cancer, ovarian cancer, testicularcancer, melanoma, lung cancer, or colon cancer. Accordingly, themammalian cell targeted by the PAP bioconjugate will be a cancer cell ora cell on the surface of a tumor blood vessel. More preferably, the cellwill be one associated with leukemia, lymphoma, a brain tumor,neuroblastoma, osteosarcoma, soft tissue sarcoma, breast cancer,prostate cancer, ovarian cancer, testicular cancer, melanoma, lungcancer, or colon cancer.

[0047] Many cancer cells overproduce cytokine receptors. Thus, thetargets for this type of therapy can be growth factor receptors,differentiation antigens, or other less characterized cell S surfaceantigens. Accordingly, effective targeting moieties include, but are notlimited to, cytokines, cytokine subunits, antibodies e.g., monoclonalantibodies, and antibody fragments and subunits e.g., monoclonalantibody fragments, single chain variable region polypeptides, andcytokines. Examples of targeting moieties include, but are not limitedto, a monoclonal antibody, monoclonal antibody fragment, or single chainvariable region polypeptide directed against the CD2, CD3, CD4, CD5,CD7, CD13, CD14, CD19, CD22, CD24, CD33, CD40, CD45, CD72, TXU.1, NXU.1,TP-1, or TP-3 antigen. Furthermore, the targeting moiety of the presentinvention may be a cytokine. If the targeting moiety is a cytokine,preferred cytokines include, but are not limited to, GM-CSF, IL-2, IL-3,IL4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, EGF, FGF, PDGF, or NGF. Seealso the '382 and the '482 patents.

[0048] Preferably, the targeting moiety will be a monoclonal antibody,monoclonal antibody fragment, or an antibody-derived single chainvariable region polypeptide, that binds to the surface of cancer cellsor tumor blood vessels. Most preferably, the targeting moiety will be amonoclonal antibody, monoclonal antibody fragment, or single chainvariable region polypeptide directed against the CD2, CD3, CD4, CD5,CD7, CD13, CD19, CD22, CD24, CD33, CD40, CD45, CD72, TXU.1, NXU.1, TP-1,or TP-3 antigen.

[0049] In some embodiments, the targeting moiety is a cytokine or singlechain variable region polypeptide derived from an antibody which doesnot bind to a receptor expressed on normal pluripotent bone marrowprogenitor cells. If the targeting moiety is a cytoline, it is preferredthat it be GM-CSF, IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-9, IL-10,IL-12, EGF, FGF, PDGF, or NGF. If the targeting moiety is a single chainvariable region polypeptide, it is preferred that it be directed againstthe CD2, CD3, CD4, CD5, CD7, CD13, CD19, CD22, CD24, CD33, CD40, CD45,CD72, IXU.1, NXU.1, TP-1, or TP-3 antigen. More preferably, the singlechain variable region polypeptide is the Fab fragment, e.g., of antibodyB43. B43 is a murine IgG1, alpha.monoclonal antibody (MoAb) recognizinga 95 kDa target B lineage restricted phosphoglycoprotein, which isidentified as the CD19 antigen according to the World HealthOrganization (WHO) established CD (cluster of differentiation)nomenclature. The chemical, immunological and biological features of B43MoAb have been described in detail in previously published reports.Uckun et al., Blood, 71:13 (1988).

[0050] The bioconjugates of the present invention may also be used forthe treatment of AIDS. In these embodiments, it is preferred that thetargeting moiety be a monoclonal antibody, monoclonal antibody fragment,or antibody-derived single chain variable region polypeptide that bindsto the surface of T-cells or monocytes or macrophages. Most preferably,the targeting moiety will be a monoclonal antibody, monoclonal antibodyfragment, or single chain variable region polypeptide directed againstthe CD2, CD3, CD4, CD5, CD7, CD14 or TXU.1 antigen. In embodiments wherethe bioconjugate is in the form of a fusion protein, it is preferredthat the targeting moiety be a cytokine that binds to the surface ofT-cells or monocytes or macrophages. Most preferably, the targetingmoiety will be M-CSF, GM-CSF, IL-2, IL-3, IL4, IL-6, IL-7, IL-8, IL-9,IL-10, or IL-12.

[0051] The bioconjugates of the present invention also provide the basisfor an effective method to inhibit other retroviruses (HTLV-1, etc.) andviruses other than retroviruses including, but not limited to, membersof the herpes virus group (HSV, CMV, EBV), influenza viruses,rhinoviruses, papovaviruses (human papilloma), adenoviruses, hepatitisvirus, and the like, and diseases associated therewith. Thebioconjugates may also be useful in the treatment of autoimmune diseasescharacterized by proliferations of unwanted cells such as T-cells orB-cells. See, U.S. Pat. No. 5,011,684.

[0052] The present bioconjugates can be formulated as pharmaceuticalcompositions and administered to a human or other mammal afflicted witha condition treatable by these agents, alone or in combination in a unitdosage form comprising an effective amount of one or more of theseagents in combination with a pharmaceutically acceptable carrier orvehicle. It is preferred that the bioconjugates be parenterallyadministered, i.e., intravenously, or subcutaneously by infusion orinjection. Solutions or suspensions of the bioconjugates can be preparedin water, or isotonic saline, such as PBS, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, DMA, vegetable oils, triacetin, andmixtures thereof. Under ordinary conditions of storage and use, thesepreparations may contain a preservative to prevent the growth ofmicroorganisms.

[0053] Additionally, more specific delivery of the bioconjugates to thelungs may be accomplished via aerosol delivery systems. Thepharmaceutical dosage form suitable for aerosol delivery can includeadipot formulations such as a liposome of suitable size.

[0054] The pharmaceutical dosage form suitable for injection or infusionuse can include sterile aqueous solutions or dispersions or sterilepowders comprising the bioconjugates which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions. In all cases, the ultimate dosage form must be sterile,fluid and stable under the conditions of manufacture and storage. Theliquid carrier or vehicle can be a solvent or liquid dispersion mediumcomprising, for example, water, ethanol, a polyol (for example,glycerol, propylene glycol, and liquid polyethylene glycols, and thelike), vegetable oils, nontoxic glycerol esters, lipids (for example,dimyristoyl phosphatidyl choline) and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the formation ofliposomes, by the maintenance of the required particle size in the caseof dispersion or by the use of nontoxic surfactants. The prevention ofthe action of microorganisms can be accomplished by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars, buffers or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the inclusion in thecompositions of agents delaying absorption, for example, aluminummonostearate hydrogels and gelatin.

[0055] Sterile injectable or infusable solutions are prepared byincorporating the bioconjugates in the required amount in theappropriate solvent with various of the other ingredients enumeratedabove, and as required, followed by filter sterilization. In the case ofsterile powders for the preparation of sterile injectable or infusablesolutions, the preferred methods of preparation are vacuum drying andthe freeze drying techniques, which yield a powder of the activeingredient plus any additional desired ingredient present in thepreviously sterile-filtered solutions.

[0056] Furthermore, suitable formulations for the bioconjugates of thepresent invention include those suitable for oral, rectal, nasal,topical (including, ocular, and sublingual) or vaginal administration orin a form suitable for administration by inhalation or insufflation. Theformulations may be prepared by any of the methods well known in the artof pharmacy.

[0057] Such methods include the step of bringing into association thebiotherapeutic agent with liquid carriers or finely divided solidcarriers or both and then, if necessary, shaping the product into thedesired formulation.

[0058] Pharmaceutical formulations suitable for oral administration mayconveniently be presented as discrete units such as capsules, sachets,or tablets, each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution, a suspension or asan emulsion. The active ingredient may also be presented as a bolus,electuary or paste. Tablets and capsules for oral administration maycontain conventional excipients such as binding agents, fillers,lubricants, disintegrants, or wetting agents. The tablets may be coatedaccording to methods well known in the art. Oral liquid preparations maybe in the form of, for example, aqueous or oily suspensions, solutions,emulsions, syrups or elixirs, or may be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations may contain conventional additives such assuspending agents, emulsifying agents, non-aqueous vehicles (which mayinclude edible oils), or preservatives. The bioconjugates may also beformulated for intra-nasal or ocular administration. In this form ofadministration, the active ingredient may be used as a liquid spray ordispersible powder or in the form of drops. Drops, for example, eyedrops, may be formulated with an aqueous or non-aqueous base alsocomprising one or more dispersing agents, solubilizing agents orsuspending agents. Liquid sprays are conveniently delivered frompressurized packs.

[0059] For administration by inhalation, the bioconjugates areconveniently delivered from an insufflator, nebulizer or a pressurizedpack or other convenient means of delivering an aerosol spray.Pressurized packs may comprise a suitable propellant such asdichlorodifluoromethane, trichlorofluromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount.

[0060] Alternatively, for administration by inhalation of insufflation,the bioconjugates may take the form of a dry powder composition, forexample, a powder mix of the compound or a suitable powder base such aslactose or starch. The powder composition may be presented in unitdosage form in, for example, capsules or cartridge or e.g., gelatin orblister packs from which the powder may be administered with the aid ofan inhaler of insufflator.

[0061] Additionally, the bioconjugates as well as the free recombinantPAP are well suited to formulation or controlled release dosage forms.The formulations can be so constituted that they release the active dryingredient only or preferably in a particular physiological location,optionally over a period of time. The coatings, envelopes, andprotective matrices may be made, for example, from polymeric substancesor waxes. The compounds can also be delivered via patches fortransdermal delivery, subcutaneous implants, infusion pumps or viarelease from implanted depot sustained release dosage forms.

[0062] The dosage of the bioconjugates in said composition can be variedwidely, in accord with the size, age and condition of the patient andthe target cancer. Based on animal data, it is expected that the dosagecan be varied between 0.025 mg/kg/day and 1 mg/kg/day, administered overa period of about 3 to 5 days. Other details regarding how to prepareand administer the bioconjugates may be found in U.S. Pat. No.6,146,628.

[0063] The present invention will now be further described in the courseof the following examples. They are merely illustrative of workunderlying the invention or of various embodiments of the invention, andthus should not be construed as limiting in any way.

EXAMPLE 1

[0064] Induction of PAP expression in yeast is known to have a cytotoxiceffect (1). This cytotoxicity was presumed to be a result of translationinhibition due to depurinated rRNA. This example provides evidence thatPAP depurination can occur in the absence of cytotoxicity and that bothevents are independent. It also describes a nontoxic mutant form of PAPthat retains full antiviral activity and is expressed to significantlyhigh levels in both plants and yeast host systems. The mutants wereisolated by engineering several mutations into specific regions of thePAP gene. Following the mutagenesis, the resulting clones were screenedfor toxicity, viability, as well as depurination activity in yeast.Several of these isolated mutants (as shown in Table 1) were found todepurinate ribosomes in the absence of toxicity. One of these isolatedproteins was studied in tobacco plants to demonstrate the presence andmagnitude of antiviral activity.

[0065] Plasmids

[0066] Construction of the mutant PAPY123A

[0067] The tyrosine codon at position 123 in pMON8588 (PAP-wild type(wt) in pGEM) was mutated to an alanine codon (TAT to GCT) using theQuikChange site-directed mutagenesis kit (Stratagene). PAPY123A wascloned into the yeast vector pNT198 containing a GAL1 promoter, PGK1polyadenylation signal, and LEU2 auxotrophic marker resulting in pNT242.PAPY123A was also cloned into the plant expression vector pMON977resulting in pNT220.

[0068] Construction of the Mutant PAPL251*

[0069] The leucine codon at position 251 in pMON8588 was mutated to astop codon (CTC to TAG) using site-directed mutagenesis as describedabove. PAPL251* (i.e., PAP (1-250)) was cloned into the yeast vectorpNT198 resulting in pNT347.

[0070] Strains and Media

[0071]Saccharomyces cerevisiae PSY1 (MATa ade2-1 trp1-1 ura3-1leu2-3,112 his3-11,15 can1-100) was used for all of the yeast assays.Synthetic complete medium H lacking leucine [H-leu]) with 2% galactoseor raffinose was used for controlling the induction of the PAP gene fromthe pNT242 and pNT347 plasmid vectors.

[0072] Induction of PAP Mutants in Yeast and Growth Curve

[0073] The PAP mutants or PAPwt were expressed in yeast from thegalactose-inducible GAL1 promoter. Yeast were first grown in H-leucontaining 2% raffinose to an OD₆₀₀ of 0.6. The cells were brieflypelleted at 1500g, washed once with H-leu, and resuspended at an OD₆₀₀of 0.6 in 200 ml of H-leu containing 2% galactose for up to 10 hours. 25ml and 5 ml were sampled from this culture every 2 hours for RNA andprotein isolation, respectively. OD₆₀₀ readings were taken every hour toassess growth rates and toxicity.

[0074] Immunoblot Analyses

[0075] For analysis of protein expression in yeast, PAPY123A or PAPL251*were expressed in yeast as described above. Protein was extracted usingglass beads and Buffer A (25 μM Tris pH 7.5, 1 μM EGTA, 10 μMβ-glycerophosphate, 0.1 μM Na₃VO₄, 1 μM DTT, 5% glycerol, 1 μM PMSF) andquantified using the Bio-Rad Protein Assay reagent. 7.5 μg of totalprotein were separated with 12.5% SDS-PAGE and transferred tonitrocellulose as described previously (2). The blots were probed withanti-PAP antibody (1:500) overnight at 4° C. The blots were laterstripped for 30 minutes with 8M guanidine HCl prior to reprobe for equalloading with anti-G6PD (1:5000). For analysis of protein expressionplants, PAPY123A was expressed and extracted from plants as describedpreviously for other transgenic plants (3). Briefly, 3 to 4 leaf discswere ground in a 1.5 ml tube in the presence of Buffer A plus plantprotease inhibitors (Sigma). Forty (40) μg of crude extract were loadedas above and probed with PAP antibody (1:2000).

[0076] Depurination Analyses

[0077] Total cellular RNA was extracted as previously described (4). One(1) ug of total RNA (7.5 μg in the case of PAPL251* was additionallytested) was used for primer extension analysis to assay the depurinationstate of the ribosomal RNA at each of the time points described above.Primer extension was carried out using AMV reverse transcriptase(Promega) following the protocol provided by the manufacturer with thechanges described here. The ribosomal primer (described in 4) wasend-labeled with ³²P γ-ATP utilizing T4 kinase (Gibco-BRL).Approximately 1×10⁶ cpm (2 ng) of labeled primer was hybridized to totalcellular RNA for 15 minutes at 70° C. then 1 hour at 43° C. in formamidebuffer (40 mM PIPES, 1 mM EDTA, 0.4M NaCl, 80% deionized formamide). TheRNA was then precipitated with 10%1 g of tRNA and resuspended in AMV-RTbuffer and extended at 43° C. for 1 hour in a volume of 10 μl. Thereaction was stopped with addition of 10 μl of stop solution (USB) and 5μl was analyzed on a 6% denaturing urea-acrylamide gel. The site ofribosomal depurination was subcloned into a vector and sequenced toprovide a size marker. The intensity of the depurination was quantifiedwith a phosphoimager and normalized to a higher MW extension product.Measurement of depurination of ribosomes by PAP proteins, using primerextension, is described in Hudak et al., RNA 6:369-380 (2000).

[0078] Antiviral Studies of PAPY123A in Plants

[0079] pNT220 was transformed into ABI Agrobacterium and geneticallyengineered into Nicotiana tabacum NN plants via anAgrobacterium-mediated transformation procedure previously described inLodge et al., PNAS 90:7089-7093 (1993). The R1 transgenic plants wereinoculated with tobacco mosaic virus (TMV) at the concentration of 2μg/ml to test for virus resistance (Lodge et al.). Local lesion numberscaused by TMV infection of inoculated leaves were counted and comparedto non-transgenic wilt-type plants. pNT220 was also geneticallyengineered into N. tabacum nn plants as described above. The R1transgenic plants were inoculated with potato virus X (PV at theconcentration of 10 μg/ml to test for virus resistance (Lodge et al.)Local lesion numbers caused by PVX infection of inoculated leaves werecounted and compared to non-transgenic wild-type plants.

[0080] Results

[0081] Growth Curves

[0082] The results are graphically illustrated in FIG. 1. While theexpression of PAPwt in yeast was toxic, the expression of both PAPY123Aand PAPL251* were nontoxic.

[0083] Immunoblot Analysis

[0084] Yeast

[0085] Immunoblot analysis for PAPwt indicated that protein was producedat 2 hours post-induction and increased a slight bit to reach a plateauof expression by 10 hours post-induction. Results for PAPY123A showedprotein expression at 2 hours to be similar to PAPwt but the abundanceincreased exponentially to 10 hours. PAPL251* showed abundant protein at2 hours post-induction (greater than both PAPwt and PAPY123A), butreached a plateau soon after. The normalization of PAP protein to G6PDprotein in all three cases confirmed equal protein loading.

[0086] Plants

[0087] Immunoblot analysis for PAPY123A in transgenic plant lines showedabundant protein expression as compared with PAPx and PAPv, two othermutant forms of PAP.

[0088] Depurination Activity

[0089] The results of PAPwt depurination graphically illustrated in FIG.2A indicate that PAP can depurinate ribosomes by 2 hours post-inductionand that maximal depurination occurs at 4 hours post-induction. Aftermaximal depurination, the relative amounts of depurinated rRNAdecreases.

[0090] The results of PAPY123A depurination graphically illustrated inFIG. 2B indicate that the mutant protein depurinates but does so after alag period. PAPY123A depurination of rRNA begins to surpass PAPwtdepurination at 8 hours-post-induction and is quite activelydepurinating ribosomes at 10 hours post-induction. This is verydifferent from what is seen by PAPwt.

[0091] The results of PAPL251* depurination graphically illustrated inFIG. 2C indicate that the mutant protein depurinates but at a greatlyreduced rate. In fact, higher starting amounts of total cellular RNAwere required to appreciate the levels of depurination. PAPL251*depurination of rRNA remains below levels of PAPwt depurination at allhours-post-induction. Again, this is very different from what is seen byPAPwt. This graph is a composite of two experiments, one using 1 μg andthe other using 7.5 μg of total cellular RNA as starting material.

[0092] Antiviral Studies of PAPY123A in Plants

[0093] As Table 1 demonstrates, the local lesion numbers caused by TMVinfection on the inoculated leaves of the NT220-transgenic plants weresignificantly lower to the numbers on the non-transgenic wild-type (wt)plants. TABLE 1 Resistance of transgenic tobacco NN plants (PAPY123A) toinfection by TMV Local Lesion # Local Lesion # Line # Experiment 1Experiment 2 NT220-5-1 23 40 NT220-5-2 12 12 NT220-5-3 14 8 NT220-5-4 1130 NT220-5-5 4 20 NT220-5-6 12 31 NT220-5-7 30 5 NT220-5-8 5 20NT220-5-9 16 45 NT220-5-10 9 24 Avg. = 13.6 ± 7.9 Avg. = 23.5 ± 15.2 wt1130 120 wt2 120 150 wt3 150 150 wt4 140 160 wt5 150 130 wt6 120 150 wt7120 160 wt8 150 140 wt9 120 120 wt10 150 130 Avg. = 135 ± 14.3 Avg. =141 ± 15.2

[0094] The data in Table 2 demonstrate that the local lesion numberscaused by PVX infection on the inoculated leaves of the NT220-transgenicplants were significantly lower than the numbers on the non-transgenicwild-type (wt) plants. TABLE 2 Resistance of transgenic tobacco nnplants (PAPY123A) to infection by PVX Line # Local Lesion # NT220-2-1 40NT220-2-2 35 NT220-2-3 45 NT220-2-4 40 NT220-2-5 47 NT220-2-6 60NT220-2-7 38 NT220-2-8 60 Avg. = 45.6 ± 9.6 wt1 80 wt2 90 wt3 75 wt4 60wt5 90 wt6 95 wt7 80 wt8 70 Avg. = 80 ± 11.6

[0095] Discussion

[0096] This is believed to be the first demonstration of a mutant formof PAP which allows cells expressing the mutant protein to continuegrowing normally in the face of depurination.

[0097] The expression profiles indicate that an abundant amount ofprotein was produced in PAPY123A and that this protein was responsiblefor depurination. The observation that PAPY123A depurinates ribosomesonly when the mutant PAP is expressed at very high levels might indicatethat this is a concentration effect. That is, once the concentration ofPAP reaches a certain level, a threshold for depurination is breachedand the effects on rRNA can be seen. In the case of PAPL251*, proteinexpression is subdued as is depurination yet protein expressioncontinues above PAPwt levels while depurination is steady at levelsbelow PAPwt.

[0098] 1. Hur et al., Proc. Natl. Acad. Sci. U.S.A. 92:8448 (1995)

[0099] 2. Hudak et al., J. Biol. Chem 274:3859 (1999)

[0100] 3. Lodge et al., Proc. Natl. Acad. Sci. U.S.A. 90:7089 (1993)

[0101] 4. Cui et al., EMBO J. 15:5726 (1996)

EXAMPLE 2

[0102] PAPY123A does not autoregulate the accumulation of its own mRNATo measure the effect of PAPwt and PAPY123A on accumulation of its mRNA,Yeast cells, containing NT242 were harvested at various times afterinduction by galactose, and the level of PAP mRNA was measured by RNaseprotection assay. A 252 nt [³²P]-labeled minus-strand RNA correspondingto the 3′ end of PAP mRNA was transcribed and hybridized in the presenceof excess probe with total RNA extracted from cells harboring PAPwt andPAPY123A plasmids. A 281 nt [³²P]-labeled minus-strand CYH2 RNA, whichencodes the constitutively expressed ribosomal protein L29, served asthe internal loading control (Fried et al., Nucleic Acids Res.10:3133-3148 (1982)). Samples were electrophoretically separated and theintensities of the protected bands were quantified using aPhosphoImager. The ratios for signals from the PAPwt or PAPY123A mRNAsto the CYH2 mRNA were used as relative measures of the steady-stateabundance of the PAPwt and PAPY123A mRNAs. As the expression of PAPwtwas induced, the level of PAPwt mRNA decreased dramatically relative tothe CYH2 mRNA. See FIG. 3A. At ten hours post-induction, PAPwt mRNAlevels had decreased to about 10% of the levels observed at two hourspost-induction. The mutant PAPY123A did not manifest such a decrease inthe level of its mRNA and quite contrary to PAPwt, the mRNA levelsbetween two and ten hours were quite similar. See FIG. 3B. Accordingly,this PAP mutant of the present invention can be produced in hosts atrelatively high levels compared to wild-type PAP.

EXAMPLE 3

[0103] To examine the relationship between ribosome depurination andmRNA destabilization in cells expressing PAP, a highly sensitive primerextension analysis was used to examine the extent of ribosomedepurination and mRNA turnover at different times after induction of PAPexpression. PAP mRNA levels were quantified by RNAse protection analysisusing the U3 RNA as the internal control. Quantification of the level ofdepurination in the RNA samples from wild type PAP (NT188) and L71R PAP(NT538) indicated that depurination was detected two hours after PAPinduction and maximal depurination of rRNA in the cell occurred bybetween 2-4 hours after induction (FIG. 4). RNase protection analysis ofmRNA levels indicated that wild type PAP mRNA levels increased up tofour hours on galactose (FIG. 5B). PAP mRNA was destabilized after 4hours even when transcription was not repressed, indicating that therate of RNA degradation exceeded the rate of RNA synthesis (FIG. 5B).Ribosome depurination decreased slightly after four hours, while PAPmRNA levels decreased dramatically. These results indicate that rRNA canbe depurinated in conditions when PAP mRNA is not degraded. This isconsistent with previous results, which indicate that low levels of PAPpresent after transcription and translation shut-off can depurinate therRNA in trans. The depurination of rRNA could precede thedestabilization of mRNA or the two events could be independent.

[0104] To determine if rRNA depurination could be separated from mRNAdestabilization, a nontoxic PAP mutant, NT538 (L71R) which depurinatesribosomes was tested. By 4 hours post induction, similar levels ofdepurination are observed in NT188 and NT538 (FIG. 4) cells. However,after 4 hours, the depurination in cells containing NT538 increases tomuch higher levels than in NT188 (FIG. 4). RNase protection analysis ofPAP mRNA levels in NT538 cells indicated that PAP mRNA is notdestabilized in NT538 cells after 4 hours of induction even though theribosomes are depurinated at higher levels than in NT188 cells. (FIG.5A) These results indicate that the activity of PAP on rRNA can bedissociated from its effects on mRNA stability.

[0105] To examine the impact of rRNA depurination and mRNAdestabilization on cell viability, cells containing NT188 or NT538plasmid were grown on liquid H-Leu media containing galactose anddifferent dilutions were plated on H-Leu plates containing glucose. Thecolony forming units were counted based on the dilution analysis. Asshown in FIGS. 6A and 6B, the results of the viability analysisindicated that cell viability decreases logarithmically in PAPexpressing cells up to 10 hours post induction. In contrast, there isvery little decrease in cell viability L71R PAP expressing cells. At 10hours post-induction, NT538 (PAP-L71R) was only reduced in viability by0.9 log or about 9-fold whereas NT188 (PAPwt) was reduced in viabilityby 3.3 log or about 2000-fold as compared with NT224 (PAPx). Theseresults suggest that the decrease observed in cell viability does notcorrelate with rRNA depurination or inhibition of translation, but doescorrelate with mRNA destabilization. Therefore, viability of cellsexpressing PAP L71R is not significantly reduced because this mutantdoes not affect mRNA stability, even though it depurinates mRNA. Theseresults indicated that inhibition of growth does not necessarily lead toinhibition of cell viability because cells containing NT538 areinhibited in growth, but not viability. As shown in FIG. 6C, growth onliquid media correlates with the ability to depurinate rRNA and inhibittranslation, but not viability.

EXAMPLE 4

[0106] Isolation and Characterization of Pokeweed Antiviral ProteinMutations in Saccharomyces cerevisiae: Identification of ResiduesImportant for Cytotoxicity and Depurination of rRNA

[0107] To identify residues critical for cytotoxicity of PAP, systematicdeletions were made from the 5′ and the 3′ ends of the PAP cDNA.Cytotoxicity and the ability of the mutant proteins to depurinate yeastribosomes in vivo were examined.

[0108] Results of these assays demonstrated that truncating the first 16amino acids of PAP by introducing a Met in place of Tyr at position 16(Y16M) eliminated the cytotoxicity of PAP and its ability to depurinateribosomes. Point mutations at Y16 (Y16A or Y16F) did not inhibitcytotoxicity or depurination of ribosomes, indicating that Y16 alone isnot responsible for cytotoxicity. Deletion of the first 13 amino acidsof PAP, by introducing a Met in place of Ser14 did not affect thecytotoxicity of PAP or its depurination ability. However, combination ofthe S14M mutation with a mutation in Y16 (Y16A) resulted in a nontoxicprotein, which depurinated ribosomes, indicating that Y16- and S14 arecritical for cytotoxicity, but not depurination of ribosomes. Theseresults indicate that cytotoxicity of PAP is not entirely due todepurination of ribosomes.

[0109] Deletion analysis of the C-terminal domain of PAP indicated thata nonsense codon introduced at L252 eliminated the cytotoxicity of PAP,but not its depurination activity. Mutation of Leu 252 to Lys (L252K)did not affect cytotoxicity or depurination activity, suggesting thatLeu 252 by itself is not critical for cytotoxicity. Depurinationactivity of PAP was abolished when a stop codon was introduced at R241,indicating that residues critical for ribosome depurination aredownstream of R241.

[0110] These results demonstrate that sequences responsible forcytotoxicity of PAP and its depurination activity can be separated atthe C-terminus of PAP, providing evidence that cytotoxicity of PAP isnot necessarily a direct result of depurination.

[0111] Materials and Methods

[0112] Mutations of PAP cDNA were achieved by using the StratageneQuikChange™ site-directed mutagenesis kit. Mutations were introducedinto a set of oligomeric primers that were used for PCR-amplificationsof the template plasmid pMON8588 with wild type PAP in pGEM (Promega)background by Pfu DNA polymerase. After PCR-amplifications, the templateplasmid pMON8588 was removed by DpnI digestion. The mutated plasmidswere transformed into Escherichia coli DH5. Mutant plasmids wereconfirmed by sequencing using the 17 Sequenase Version 2.0 sequencingkit (USB). cDNAs encoding PAP N-terminal mutants were subcloned as BamHI-Hind III m fragments into the yeast expression vector YEp351 underthe control of a galactose-inducible GAL1 promoter.

[0113] Yeast Transformation: Yeast cells (Saccharomyces cerevisiaestrain W303 (MAT α, ade2-1 trp1-1 ura3-1 leu2-3, 112 his3-11, 15can1-1000) were transformed according to Ausubel et al., (1994). Thetransformed yeast suspension was divided in half and half was platedonto H-leu supplemented with 2% dextrose and the other half onto H-leuwith 2% galactose.

[0114] Transformed yeast was allowed to grow for 72 hours at 30° C.Toxicity of the PAP mutants was verified by re-plating selected coloniesonto both 2% raffinose and 2% galactose.

[0115] Growth curves: Yeast transformed with either wild type or mutantforms of PAP were grown in synthetic H-leu medium supplemented with 2%raffinose at 30° C. with shaking at 240 r.p.m. in a total volume of 100ml until an A600=0.6. Yeast cells were pelleted by centrifugation at2,000×g for 5 min, washed with H-leu medium and resuspended in H-leumedium containing 2% galactose to induce the expression of PAP or PAPmutants. At zero time (immediately following induction) and at each hourfollowing induction, 1 ml aliquots were removed and A600 measured.Doubling times were calculated from the growth curves and compared tothe doubling time of PAPwt.

[0116] Yeast Protein Expression Analysis: Yeast containing cDNAs of PAPor PAP mutants were grown as described for growth curves, in a 10 mlvolume and induced with 2% galactose for 6h. Cells were pelleted bycentrifugation at 2,000×G for 5 min. Pellets were resuspended in anequal volume of cold (4° C.) Buffer X (25 mM Tris-HCl pH 7.5, 100 mMsodium vanadate, 10 mM β-glycophosphatase, 1 mM EGTA, 1 mM DTT, 1 mMPMSF, 5% glycerol) and 0.3 g of 0.5 mm diameter glass beads. Cells werevortexed for 2 min and centrifuged at 16,000×g for 5 min. Supernatanttotal protein was quantified by Bradford using BSA as a standard. Totalprotein (15 μg) was separated through 12% SDS-PAGE, transferred tonitrocellulose, and blocked by incubation with PBST (phosphate bufferedsaline with 0.1% Tween 20) in 5% nonfat milk for 2 h. Proteins wereprobed by overnight incubation with an affinity purified polyclonalantibody to PAP (1:5000) in PBST-5% milk and secondary goat anti-rabbitIgG conjugated to horseradish peroxidase (1:5000) in PBST-5% milk for1.5 h. Mutant PAP proteins were visualized by chemiluminescence using aRenaissance kit (NEN, DuPont). To confirm equal loading of totalprotein, blots were stripped with 8 M guanidine hydrochloride andreprobed with a polyclonal G6PD (1:5000; Chemicon, Temecula, Calif.) andhorseradish peroxidase conjugated secondary donkey anti-goat IgG(1:5000).

[0117] rRNA depurination assay. Yeast cells (100 ml) grown as describedfor growth curves, were harvested following a 6 h induction of PAP andPAP mutants and used to isolate ribosomes as previously described (Hudaket al., 1999). To determine if PAP mutants depurinated the S/R loop whenexpressed in vivo, primer extension analysis was preformed essentiallyas described in Hudak et al., 2001. Purified ribosomal RNAs (1 μg) wereincubated with a 5′ [32P] end-labeled oligonucleotide primer(5′-GGCGTTCAGCCATAATCC-3′) complementary to the 3′-end of yeast 25SrRNA. Primer extension was performed as described (Ioranov et al., 1997)with minor modifications. Namely, the total reaction volume was 15 μl,to which 5 μl of formamide buffer was added to stop the extensionwithout the precipitation of RNA and resulting cDNA. An aliquot of thisreaction (4 μl) was separated on a 6% polyacrylamide/7M urea gel andvisualized by autoradiography. To determine the position of rRNAdepurination, a sequencing ladder of DNA corresponding to the yeast 25SrRNA was separated on the same gel (Hudak et al., 2001).

[0118] Results And Discussion

[0119] The results are summarized in Table 3, which is appended to thisexample.

[0120] Mutations Within the N-Terminal Domain of PAP

[0121] To identify residues that are important for cytotoxicity withinthe N-terminal domain of PAP, site-directed mutagenesis was used to makesystematic deletions from the N-terminus and to introduce pointmutations at critical residues. N-terminal deletions were made byintroducing a Met codon at conserved residues and deleting the residuesupstream of Met by introducing a BglII site. Protein from all mutantswas expressed following 6 h induction in yeast, though expressionpatterns varied. Wild type PAP expressed in yeast was present in twoforms, the mature protein at 29 kDa (parallel to standard lane inimmunoblots) and a higher molecular weight form, presumably theprecursor form of PAP seen previously in yeast lysates (Hur et al.,1995; Hudak et al., 1999). The molecular weight of C-terminal deletionmutants such as NT1246 (W237*) and NT509 (L240*) were approximately 26.5kDa, indicating the expected lower size of these proteins missing either26 or 23 amino acids, respectively.

[0122] Systematic deletions were made from the N-terminus of mature wildtype PAP, from the 14^(th) to the 39^(th) amino acid. As shown in Table3, deletion of the N-terminal signal peptide and 14 amino acids from theN-terminus of mature PAP (NT418, S14M) did not affect the cytotoxicityof PAP. The doubling time of yeast expressing this mutant was 7.8 hcompared with 10.4 h for wild type PAP (NT188), which contains both theN-terminal signal sequence and the C-terminal extension. The level ofdepurination of S14M PAP was 70% of wild type PAP. Similarly,substitution of Lys with Met (NT413, K15M) resulted in a cytotoxicprotein that inhibited yeast growth and depurinated ribosomes to 79% ofwild type levels. These results indicated that deletion of theN-terminal signal sequence and the first 15 amino acids from theN-terminus of mature PAP does not affect its cytotoxicity or ability todepurinate ribosomes.

[0123] In contrast, changing Tyr16 to Met generated a deletion mutantthat was unable to depurinate ribosomes and was also noncytotoxic whenexpressed in vivo, indicating that Y16 is required for cytotoxicity.Further deletions from the N-terminus, which included T18M and deletionof the N-terminus to Met 39, resulted in nontoxic proteins that did notdepurinate ribosomes. These results indicated that either Y16 alone orY16 and another amino acid upstream are required for cytotoxicity of PAPand its ability to depurinate ribosomes. Point mutations of Y16 toeither Ala (NT321) or Phe (NT324) created a protein that regained bothcytotoxicity and depurination ability, indicating that the tyrosinealone is not entirely responsible for these characteristics, but thatamino acids upstream of this tyrosine likely contribute.

[0124] Further evidence for this was obtained when Y16A mutation wascombined with a mutation of Ser14 to Met (NT472) (Table 3) The resultingdouble mutant (S14M,Y16A) protein lost its cytotoxicity, indicating thateither Ser14 or another amino acid within the first 14 amino acids ofPAP, in combination with Tyr16, contributes to the cytotoxicity of PAP.The doubling time of cells expressing NT472 was 6.8 h, similar to thegrowth rate of the nontoxic PAP mutant NT224 (6.4 h). However, ribosomeswere depurinated in yeast expressing the double mutant (NT472),providing evidence that nbosome depurination alone does not lead tocytotoxicity. Changing the Ser to other amino acids did not result inthe same inhibition. For example, exchange of the Ser for Thr (NT450)did not affect cytotoxicity or depurination activity, as may be expectedbecause the hydroxyl group and relative size of the amino acid weremaintained. The growth rate, as measured by doubling time, of NT450 wassimilar to cells expressing wild type PAP, 10.1 h and 10.4 hrespectively, and its level of depurination was similar to wild typePAP, at 99%. Substitution of Ser for the hydrophobic amino acid Ile(NT473) or the aromatic ring amino acid Phe (NT469) resulted in toxicproteins that depurinated to levels comparable to wild type PAP.

[0125] Taken together, these results indicate that S14 alone is notresponsible for cytotoxicity and S14 together with Y16 may contribute tothe cytotoxicity of PAP. The double mutant form of PAP depurinatedribosomes to 79% of the level of wild type PAP, a level between thatobserved for the single mutations Y16A (97%) and S14M (70%). Inaddition, the S14MY16A PAP was nontoxic to cells i.e., doubling timeswere similar to yeast cells expressing PAPx, 6.8 h compared with 6.4 h,respectively. However, the cytotoxic mutant K15M depurinated ribosomesalso to 79% and doubling times of yeast expressing this mutant were 8.5h. These results indicate that nbosome depurination alone is notentirely responsible for the cytotoxicity of PAP.

[0126] Mutations Within the C-terminal Domain of PAP

[0127] Sequential deletion mutants from the 3′-terminus of PAP indicatethat changing the last amino acid of mature PAP to a stop codon anddeleting the C-terminal extension of PAP does not affect itscytotoxicity or depurination ability. The T262* PAP is cytotoxic anddepurinates ribosomes to 96% of wild type levels. The Y254* PAP is alsocytotoxic and depurinates ribosomes to the same extent as wild type PAP(100%). The N253* PAP is cytotoxic, but depurinates to 64% of wild typelevels. Cytotoxicity is lost when L252 is deleted (NT420). The L252* PAPis capable of depurinating ribosomes, albeit at lower levels than wildtype PAP (39%). Similarly, L251* PAP is not cytotoxic and depurinatesribosomes to only 35% of the wild type levels. The lack of toxicity ofboth L252* and L251* PAP may be due to their lower levels ofdepurination relative to wild type PAP (39% and 35% respectively),suggesting that a threshold level exists at which yeast cells willtolerate some degree of nbosome depurination without reduction inoverall growth rates. The relative amount of depurination measured forN253* PAP was 64%, an intermediate value between L252* measured at 39%and the toxic mutant Y254* measured at 100%. Substitution of L252 forLys (NT457) did not alter the growth rate of cells or depurinationability of PAP, indicating that Leu252 alone is not responsible forcytotoxicity. Rather, the results indicate that L252 and residuesdownstream are important determinants of cytotoxicity. This observationis supported by the sequential increase in both toxicity anddepurination observed between L252* and Y254*.

[0128] The C-terminal deletion analysis indicates that cytotoxicity ofPAP is lost before its ability to depurinate ribosomes. Depurinationceased when a stop codon was introduced at R241. These results indicatethat R241 and residues downstream are critical for ribosomedepurination. Increased length of deletion from the C-terminal endresulted in a gradual decrease in both cytotoxicity and the depurinationability of PAP. These results provide further evidence that C-terminalamino acids are critical for both toxicity and nbosome depurination. Ithas been proposed that a cleft at the interface between the central andthe C-terminal domains of PAP forms the putative substrate-binding site(Ago et al, 1994). The positively charged domain in the C-terminalregion has been proposed to provide interaction with the substrate RNA(Ago et al, 1994). Therefore, the C-terminal region of PAP may berequired for proper folding of the active site to interact with thesubstrate RNA. An alternative possibility is that the C-terminal domainmay be involved in membrane interactions prior to translocation of PAPinto the cytosol from the ER. It has been shown that efficientinternalization of transmembrane receptor proteins requires a signalsequence in the cytoplasmic tail of the protein. At least two differenttypes of internalization sequences based on either tyrosine ordi-leucine motifs have been identified in a variety of receptormolecules. Leucine-leucine or leucine-isoleucine sequence motifsimportant for internalization and/or lysosomal targeting were found inthe intercellular domains of various receptors. Thus, sequencesdownstream of R241, which include the dileucine motif in PAP, may beinvolved in membrane interactions prior to translocation of PAP into thecytosol. A point mutation in a proline residue in the same region ofricin, P250A, resulted in a marked reduction in cytotoxicity.

[0129] Mutations Within the Central Domain of PAP.

[0130] It has been proposed that amino acids Y72 and Y123 sandwich theadenine ring of nbosomal RNA in an energetically favorable stackingconformation. Subsequently, the side chain of R179 can protonate the N-3atom of the adenine base, while E176 stabilizes a positive oxocarboniumtransition state. E176 and R179 are highly conserved amino acids withinthe active site of RIPs and both participate directly in thedepurination of rRNA. As shown in Table 3, substitution of Y72 for Ala(NT241) resulted in a nontoxic protein that depurinated ribosomes tomuch lower levels than wild type PAP. The relative degree ofdepurination by Y72A PAP was 4% of wild type PAP, which confirms earlierreports that this amino acid is important for depurination of the rRNA.Replacement of Y123 with Ala (NT242) again produced a nontoxic form ofPAP, which exhibited relatively low levels of nbosome depurination (21%of wild type levels), at 6h post-induction. However, substitution ofY123 with ne (NT485) resulted in a nontoxic mutant with growth rates ofcells comparable to the active site mutant, NT224 (6.9 h and 6.4 hrespectively), but with substantially higher levels of depurination.Ribosomes from cells expressing Y123I PAP were depurinated 81% relativeto wild type PAP, without growth inhibition, providing further evidencethat cytotoxicity of PAP may not be entirely due to ribosomedepurination. Replacing Y123 with Phe (NT483) generated a toxic proteinwith doubling times of cells increased to 11.4 h. Y123F PAP depurinatedribosomes to 98% of control levels. Given that Y123I PAP was not toxicdespite 81% nbosome depurination relative to wild type PAP, whereasY123F PAP was toxic and also depurinated ribosomes, suggests thatmaintenance of the benzyl ring structure is important for toxicity ofthe conserved Y123 but not its rRNA depurination activity.

[0131] Analysis of central domain mutants indicates that there is not agood correlation between the extent of depurination and cytotoxicity,suggesting that factors other than the absolute level of depurinationmay be responsible for the cytotoxicity of PAP. Since both Y72 and Y123are thought to be involved in substrate binding, the lower level ofdepurination observed with Y72A PAP could be due to lower levels ofbinding to substrate RNA. Y72 is within the RNP2 binding domain of PAP,which appears to be critical for binding to rRNA. A previouslycharacterized mutant in this domain G75D PAP did not bind ribosomesefficiently. Other mutations constructed in the RNP2 domain (L71-Y76)indicated that all amino acids within this domain (L71, L72, V73, M74,G75 and Y76) are critical for cytotoxicity of PAP (Table 3). Incontrast, alanine substitutions at N69 (NT502), N70 (NT501) and D92(1T503) did not affect the cytotoxicity or the depurination ability ofPAP. Previous studies showed that alanine substitutions at N69, N70, F90and D92 substantially reduced the depurinating and ribosome inhibitoryactivity of PAP in vitro (Rajamohan et al., 2000). This could be due topotential differences in the localization of these proteins in vivo, allproteins in this study were expressed and characterized iii vivo.Alternatively, the assays used in the previous studies were notsensitive enough to detect low levels of depurination in vitro.

[0132] These results suggest that amino acids within the RNP2 domain(L71-Y76) of PAP are likely involved in substrate recognition. Unlikethe previously published results (Rajamohan et al., 2000), the Y123A PAPmutant was still able to depurinate ribosomes. A sensitive assay, primerextension analysis, clearly indicated that Y123A PAP depurinatedribosomes to 21% of wild type PAP. Substitution of an isoleucine residuein place of tyrosine 123 increased depurination to 81% of the controllevels, without affecting cytotoxicity. These results indicate that Y123does not play a major role in substrate binding as previously thought,but does play a major role in cytotoxicity. TABLE 3 Effect of mutationson the cytotoxicity of PAP and its ability to depurinate ribosomes.N.D.: not determined;*:stop codon) DEPURINATION MUTATION CYTOTOXICITY (%of control) DOUBLING TIME N-terminal domain Mutants NT188 Wild Type YesYes (100) 10.4 NT418 S14M Yes Yes (70.25) 7.85 NT413 K15M Yes Yes(79.44) 8.55 NT542 Y16M No No (5.95) 7.1 NT549 T18M No No (6.67) 7.3NT441 39M No No (2.05) 7.5 NT525 S14A Yes Yes (106.49) 10.2 NT426 S14EYes Yes (96.65) 10.15 NT449 S14K Yes Yes (101.01) 10.5 NT450 S14T YesYes (98.94) 10.1 NT469 S14F Yes Yes (94.33) 11 NT473 S14I Yes Yes(100.10) 9.95 NT472 S14MY16A No Yes (78.62) 6.85 NT360 K15A Yes Yes(83.46) 10.2 NT558 Y16A Yes Yes (97.11) 9.5 NT548 Y16F Yes Yes (103.86)11.0 NT569 S14AY16A Yes Yes (96.95) 9.9 Central domain Mutants NT502N69A Yes Yes (102.53) 10.5 NT501 N70A Yes Yes (95.62) 7.3 NT538 L71R NoYes (105.41) 8-9 NT241 Y72A No No (4.05) 6.5 NT532 V73E No Yes (89.59)7.5 NT533 M74R No Yes (102.6) 8.5 NT255 G75D No No (0) NT534 Y76A No Yes(98.51) 6.1 NT503 D92A Yes Yes (101.27) 10 NT242 Y123A No Yes (21.03)7.1 NT483 Y123F Yes Yes (97.84) 11.4 NT485 Y123I No Yes (80.75) 6.9NT224 E176V No No (0) 6.4 C-terminal domain Mutants NT246 W237* No No(0) NT509 L240* No No (0) 8.2 NT552 R241* No No (0) 6.2 NT510 V242* NoYes (5.42) 8.25 NT333 E244* No Yes (3.32) 6.45 NT486 A250* No Yes(42.95) 6.8 NT347 L251* No Yes (34.52) 6 NT420 L252* No Yes (38.92) 6NT456 N253* Yes Yes (64.15) 6.1 NT443 Y254* Yes Yes (100.00) 7.85 NT233T262* Yes Yes (96.32) 7.1 NT457 L252K Yes Yes (94.48) 8 NT232 C259A YesYes (97.24) 7.55 NT451 E176VW237* No No

CITATIONS

[0133] Ago et al., Eur. J. Biochem. 225:369-374 (1974).

[0134] Hudak et al., J. Biol. Chem. 274:3859-3864 (1999).

[0135] Hudak et al., RNA 6:369-380 (2000).

[0136] Hudak et al., Virology 279:292-301 (2001).

[0137] Hur et al., Proc. Natl. Acad. Sci. USA 92:8448-8452 (1995).

[0138] Rajamohan et al., J. Biol. Chem. 275:3382-3390 (2000).

INDUSTRIAL APPLICABIUIY

[0139] The present invention is useful in agricultural biotechnology aswell as in the fields of pharmaceutics and medicine.

[0140] All patent and non-patent publications cited in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains. All these publications areherein incorporated by reference to the same extent as if eachindividual publication was specifically and individually indicated to beincorporated herein by reference.

[0141] Although the invention herein has been described with referenceto particular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention.

1 3 1 1376 DNA Phytolacca americana CDS (225)..(1160) 1 ctatgaagtcgggtcaaagc atatacaggc tatgcattgt tagaaacatt gatgcctctg 60 atcccgataaacaatacaaa ttagacaata agatgacata caagtaccta aactgtgtat 120 gggggagtgaaacctcagct gctaaaaaaa cgttgtaaga aaaaaagaaa gttgtgagtt 180 aactacagggcgaaagtatt ggaactagct agtaggaagg gaag atg aag tcg atg 236 Met Lys SerMet 1 ctt gtg gtg aca ata tca ata tgg ctc att ctt gca cca act tca act284 Leu Val Val Thr Ile Ser Ile Trp Leu Ile Leu Ala Pro Thr Ser Thr 5 1015 20 tgg gct gtg aat aca atc atc tac aat gtt gga agt acc acc att agc332 Trp Ala Val Asn Thr Ile Ile Tyr Asn Val Gly Ser Thr Thr Ile Ser 2530 35 aaa tac gcc act ttt ctg aat gat ctt cgt aat gaa gcg aaa gat cca380 Lys Tyr Ala Thr Phe Leu Asn Asp Leu Arg Asn Glu Ala Lys Asp Pro 4045 50 agt tta aaa tgc tat gga ata cca atg ctg ccc aat aca aat aca aat428 Ser Leu Lys Cys Tyr Gly Ile Pro Met Leu Pro Asn Thr Asn Thr Asn 5560 65 cca aag tac gtg ttg gtt gag ctc caa ggt tca aat aaa aaa acc atc476 Pro Lys Tyr Val Leu Val Glu Leu Gln Gly Ser Asn Lys Lys Thr Ile 7075 80 aca cta atg ctg aga cga aac aat ttg tat gtg atg ggt tat tct gat524 Thr Leu Met Leu Arg Arg Asn Asn Leu Tyr Val Met Gly Tyr Ser Asp 8590 95 100 ccc ttt gaa acc aat aaa tgt cgt tac cat atc ttt aat gat atctca 572 Pro Phe Glu Thr Asn Lys Cys Arg Tyr His Ile Phe Asn Asp Ile Ser105 110 115 ggt act gaa cgc caa gat gta gag act act ctt tgc cca gcc aattct 620 Gly Thr Glu Arg Gln Asp Val Glu Thr Thr Leu Cys Pro Ala Asn Ser120 125 130 cgt gtt agt aaa aac ata aac ttt gat agt cga tat cca aca ttggaa 668 Arg Val Ser Lys Asn Ile Asn Phe Asp Ser Arg Tyr Pro Thr Leu Glu135 140 145 tca aaa gcg gga gta aaa tca aga agt cag gtc caa ctg gga attcaa 716 Ser Lys Ala Gly Val Lys Ser Arg Ser Gln Val Gln Leu Gly Ile Gln150 155 160 ata ctc gac agt aat att gga aag att tct gga gtg atg tca ttcact 764 Ile Leu Asp Ser Asn Ile Gly Lys Ile Ser Gly Val Met Ser Phe Thr165 170 175 180 gag aaa acc gaa gcc gaa ttc cta ttg gta gcc ata caa atggta tca 812 Glu Lys Thr Glu Ala Glu Phe Leu Leu Val Ala Ile Gln Met ValSer 185 190 195 gag gca gca aga ttc aag tac ata gag aat cag gtg aaa actaat ttt 860 Glu Ala Ala Arg Phe Lys Tyr Ile Glu Asn Gln Val Lys Thr AsnPhe 200 205 210 aac aga gca ttc aac cct aat ccc aaa gta ctt aat ttg caagag aca 908 Asn Arg Ala Phe Asn Pro Asn Pro Lys Val Leu Asn Leu Gln GluThr 215 220 225 tgg ggt aag att tca aca gca att cat gat gcc aag aat ggagtt tta 956 Trp Gly Lys Ile Ser Thr Ala Ile His Asp Ala Lys Asn Gly ValLeu 230 235 240 ccc aaa cct ctc gag cta gtg gat gcc agt ggt gcc aag tggata gtg 1004 Pro Lys Pro Leu Glu Leu Val Asp Ala Ser Gly Ala Lys Trp IleVal 245 250 255 260 ttg aga gtg gat gaa atc aag cct gat gta gca ctc ttaaac tac gtt 1052 Leu Arg Val Asp Glu Ile Lys Pro Asp Val Ala Leu Leu AsnTyr Val 265 270 275 ggt ggg agc tgt cag aca act tat aac caa aat gcc atgttt cct caa 1100 Gly Gly Ser Cys Gln Thr Thr Tyr Asn Gln Asn Ala Met PhePro Gln 280 285 290 ctt ata atg tct act tat tat aat tac atg gtt aat cttggt gat cta 1148 Leu Ile Met Ser Thr Tyr Tyr Asn Tyr Met Val Asn Leu GlyAsp Leu 295 300 305 ttt gaa gga ttc tgatcataaa cataataagg agtatatatatattactcca 1200 Phe Glu Gly Phe 310 actatattat aaagcttaaa taagaggccgtgttaattag tacttgttgc cttttgcttt 1260 atggtgttgt ttattatgcc ttgtatgcttgtaatattat ctagagaaca agatgtactg 1320 tgtaatagtc ttgtttgaaa taaaacttccaattatgatg caaaaaaaaa aaaaaa 1376 2 312 PRT Phytolacca americana 2 MetLys Ser Met Leu Val Val Thr Ile Ser Ile Trp Leu Ile Leu Ala 1 5 10 15Pro Thr Ser Thr Trp Ala Val Asn Thr Ile Ile Tyr Asn Val Gly Ser 20 25 30Thr Thr Ile Ser Lys Tyr Ala Thr Phe Leu Asn Asp Leu Arg Asn Glu 35 40 45Ala Lys Asp Pro Ser Leu Lys Cys Tyr Gly Ile Pro Met Leu Pro Asn 50 55 60Thr Asn Thr Asn Pro Lys Tyr Val Leu Val Glu Leu Gln Gly Ser Asn 65 70 7580 Lys Lys Thr Ile Thr Leu Met Leu Arg Arg Asn Asn Leu Tyr Val Met 85 9095 Gly Tyr Ser Asp Pro Phe Glu Thr Asn Lys Cys Arg Tyr His Ile Phe 100105 110 Asn Asp Ile Ser Gly Thr Glu Arg Gln Asp Val Glu Thr Thr Leu Cys115 120 125 Pro Ala Asn Ser Arg Val Ser Lys Asn Ile Asn Phe Asp Ser ArgTyr 130 135 140 Pro Thr Leu Glu Ser Lys Ala Gly Val Lys Ser Arg Ser GlnVal Gln 145 150 155 160 Leu Gly Ile Gln Ile Leu Asp Ser Asn Ile Gly LysIle Ser Gly Val 165 170 175 Met Ser Phe Thr Glu Lys Thr Glu Ala Glu PheLeu Leu Val Ala Ile 180 185 190 Gln Met Val Ser Glu Ala Ala Arg Phe LysTyr Ile Glu Asn Gln Val 195 200 205 Lys Thr Asn Phe Asn Arg Ala Phe AsnPro Asn Pro Lys Val Leu Asn 210 215 220 Leu Gln Glu Thr Trp Gly Lys IleSer Thr Ala Ile His Asp Ala Lys 225 230 235 240 Asn Gly Val Leu Pro LysPro Leu Glu Leu Val Asp Ala Ser Gly Ala 245 250 255 Lys Trp Ile Val LeuArg Val Asp Glu Ile Lys Pro Asp Val Ala Leu 260 265 270 Leu Asn Tyr ValGly Gly Ser Cys Gln Thr Thr Tyr Asn Gln Asn Ala 275 280 285 Met Phe ProGln Leu Ile Met Ser Thr Tyr Tyr Asn Tyr Met Val Asn 290 295 300 Leu GlyAsp Leu Phe Glu Gly Phe 305 310 3 18 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 3 ggcgttcagc cataatcc 18

1. A Pokeweed Antiviral Protein (PAP) mutant that is less toxic thanwild-type PAP and which exhibits ribosome depurination activity.
 2. ThePAP mutant of claim 1 which comprises PAP (1-262, Y123A).
 3. The PAPmutant of claim 1 which comprises PAP (1-262, S14M, Y16A).
 4. The PAPmutant of claim 1 which comprises PAP (1-262, L71R).
 5. The PAP mutantof claim 1 which comprises PAP (1-262, V73E).
 6. The PAP mutant of claim1 which comprises PAP (1-262, M74R).
 7. The PAP mutant of claim 1 whichcomprises PAP (1-262, Y76A).
 8. The PAP mutant of claim 1 whichcomprises PAP (1-262, Y1231).
 9. The PAP mutant of claim 1 which differsfrom wild-type PAP substantially in that it is truncated at itsC-terminus from 10 to 20 mature PAP amino acids.
 10. The PAP mutant ofclaim 9 which is selected from the group of PAP mutants consisting ofPAP (1-242), PAP (1-243), PAP (1-244), PAP (1-245), PAP (1-246), PAP(1-247), PAP (1-248), PAP (1-249), PAP (1-250) and PAP (1-251).
 11. ThePAP mutant of claim 1 further comprising N-terminal signal sequence ofwild-type PAP.
 12. The PAP mutant of claim 1 further comprisingC-terminal extension of wild-type PAP.
 13. A PAP mutant that is lesstoxic than wild-type PAP and which exhibits ribosome depurinationactivity, wherein said mutant is an N-terminal domain mutant.
 14. A PAPmutant that is less toxic than wild-type PAP and which exhibits ribosomedepurination activity, wherein said mutant is a central domain mutant.15. A fusion protein comprising the PAP mutant of claim 1 and atargeting moiety that binds a cell surface receptor.
 16. Animmunoconjugate comprising the PAP mutant of claim 1 and a targetingmoiety that binds a cell surface receptor.
 17. A DNA molecule encodingthe fusion protein of claim
 15. 18. A DNA molecule encoding the PAPmutant of claim
 1. 19. A chimeric DNA molecule comprising the DNAmolecule of claim 17 or claim 18 operably linked to a promoter.
 20. Thechimeric DNA molecule of claim 19 wherein said promoter is functional ina yeast cell.
 21. The chimeric DNA molecule of claim 19 wherein saidpromoter is functional in a plant cell.
 22. The chimeric DNA molecule ofclaim 19 wherein the promoter is functional in an animal cell.
 23. Thechimeric DNA molecule of claim 19 wherein said promoter is an induciblepromoter.
 24. The chimeric DNA molecule of claim 19 wherein saidpromoter is a constitutive promoter.
 25. The DNA molecule of claim 19wherein said PAP mutant comprises the N-terminal signal sequence ofwild-type PAP.
 26. The DNA molecule of claim 19 wherein said PAP mutantcomprises the C-terminal extension of wild-type PAP.
 27. A recombinantvector comprising the DNA molecule of claim 17 or claim
 18. 28. Aprotoplast stably transformed with the DNA molecule of claim 17 or claim18.
 29. A non-human host transformed with the chimeric DNA molecule ofclaim 17 or claim
 18. 30. The host of claim 29 which is an E. coli cell.31. The host of claim 29 which is a yeast cell.
 32. The yeast cell ofclaim 29 which is a Saccharomyces cerevisiae cell.
 33. The host of claim29 which is a plant cell.
 34. The host of claim 29 wherein said DNAmolecule is operably linked to an inducible promoter functional in saidhost.
 35. A transgenic plant regenerated from the protoplast of claim28.
 36. A transgenic plant comprising the chimeric DNA molecule of claim17 or claim 18, wherein the DNA molecule is expressed in said plant. 37.The transgenic plant of claim 35 or claim 36 which is a monocot plant.38. The transgenic plant of claim 37 wherein said monocot is a cerealcrop plant.
 39. The transgenic plant of claim 35 or claim 36 which is adicot plant.
 40. Seed derived from the transgenic plant of claim 35 or36.
 41. A method of making a plant that has increased resistance toviruses and/or fungi, comprising preparing a plant having a genome thatcontains the DNA molecule of claim 18 wherein said sequence isexpressed.
 42. The method of claim 41 comprising transforming aprotoplast with the DNA molecule, and generating the transgenic plantfrom the transformed protoplast.
 43. The method of claim 41 comprisingintroducing the DNA molecule into plant tissue, and regenerating theplant from the plant tissue containing the DNA molecule.
 44. Apharmaceutical composition comprising the fusion protein of claim 15 orthe immunoconjugate of claim 16 and a pharmaceutically acceptablecarrier.
 45. A method of treating a mammal suffering from cancercomprising administering the composition of claim 44 to the mammal. 46.A method of treating a mammal suffering from AIDS comprisingadministering the composition of claim 44 to the mammal.